The homology between F16 and Ser recombinases has interesting evolutionary implications. The CRV homolog of F16 is the first likely Ser recombinase to have been detected in any eukaryote or eukaryotic virus (Afonso et al., 2006
). Thus, the most parsimonious evolutionary scenario involves acquisition of a Ser recombinase gene by the ancestor of chordopoxviruses from a transposon or a bacteriophage. This scenario is reminiscent of other probable bacterial or bacteriophage contributions to the evolution of poxviruses, in particular, the Holiday junction resolvase A22 (Garcia et al., 2000
) and the predicted deubiquitinase G6 (Senkevich et al., 2008
) as well as the primase-helicase D5 and the DNA-packaging ATPase A32 that are shared with other nucleocytoplasmic large DNA viruses (Koonin and Yutin, 2010
). The subsequent evolution of chordopoxviruses apparently involved the abrogation of the recombinase activity of F16, as evidenced by the substitution of key amino acids in the active site region. This loss of activity occurred after the divergence of CRV [the deepest chordopoxvirus branch (Afonso et al., 2006
; Koonin and Yutin, 2010
)] from the common ancestor with the other chordopoxviruses but prior to the radiation of the latter, and the complete loss of this gene in the avipoxvirus branch. The loss of recombinase activity was apparently accompanied by an overall acceleration of evolution and apparently a change of function.
Assuming the latter scenario, what is the acquired new function of the poxvirus F16 homologs? Given its conservation and early expression shown here, a role in viral DNA replication or recombination seemed one possibility. However, knockout of the F16L gene had no measurable effect on virus growth in any of the many tested cell cultures including resting primary HFFs. Another gene with similar biological features is the G6R gene that is conserved in all poxviruses and encodes a putative deubiquitinating enzyme (Senkevich et al., 2008
). Knockout of the G6R gene has no effect on virus growth in all tested cell cultures and leads only to a slight attenuation in intranasal mouse infection. Conservation of these genes in most poxvirus genera suggests that their functions are not directly involved in the virus growth cycle but may be involved in generic aspects of virus-host interaction as opposed to interactions between viruses and their specific hosts.
The targeting of the F16 protein to the nucleoli, whether expressed by VACV or by transfection in uninfected cells, was unexpected. However, the myxoma virus ankyrin repeat protein M148R, which also lacks a defined nucleolar localization motif, has been reported to localize in the nucleolus (Blanie et al., 2009
). Deletion of M148R does not perturb replication in vitro but reduces virulence in a rabbit model. Another VACV protein, E3L, has been detected in the nuclei of infected cells but its nuclear function (if any) has not been characterized (Yuwen et al., 1993
). Although F16 had a 3xflag tag, the latter is unlikely to cause nuclear or nucleolar targeting as the VACV proteins I3 and B1, containing a C-terminal 3xflag tag and expressed from a transfected plasmid under the control of the CMV promoter, were analyzed in parallel with F16-3xflag and no nucleolar or nuclear membrane staining was detected (TGS, unpublished). Moreover, the 3xflag has been used in numerous studies of subcellular localization of proteins, and no specific effect on nuclear and/or nucleolar targeting has been reported. In particular, the 3xflag tag was used in a genome wide screen of herpesviruses for protein subcellular localization, with no specific effects on protein targeting (Salsman et al., 2008
The amino acid sequence of F16 does not contain known nuclear or nucleoli targeting signals (Emmott and Hiscox, 2009
) and our studies indicated that both the N- and C-terminal regions of F16 are required for localization to nucleoli. A likely possibility is that F16 targets the nucleus and nucleoli by interacting with a cellular protein. Thus far, we have been unable to detect an interaction of F16 with cellular nucleolin or nucleophosmin by coimmunopurification (TGS, unpublished) but interactions with other cellular proteins remain a possibility. We did find that F16 interacts with itself to form homodimers or higher order multimers. If there is competition between F16 binding to itself and to a cell protein, this could explain the inverse correlation of nucleolar targeting with the level of expression and confluency of cells. Conditions in which the concentration of F16 is increased (high expression) or the cell protein is decreased (confluency), might favor multimer formation and cytoplasmic localization.
Targeting of viral proteins to nucleoli is a frequent theme in the study of virus-host interactions. For example, a genome-wide screen of three distinct herpesviruses has shown that at least 12 herpesvirus-encoded proteins specifically localize to the nucleolus (Salsman et al., 2008
). Targeting of viral proteins to nucleoli has been described not only for DNA viruses such as herpesviruses, adenoviruses, and papovaviruses, and RNA viruses, such as retroviruses and influenza, that possess a defined nuclear stage of reproduction, but also for proteins of RNA viruses without any nuclear stage such as coronaviruses, arteriviruses and filoviruses as well as several groups of RNA viruses of plants (Hiscox, 2007
; Hiscox et al., 2010
; Taliansky et al., 2010
). Concomitantly, it has become evident than nucleoli are not only the factories of ribosome biosynthesis, as previously thought, but multifunctional and dynamic structures that contain hundreds of cellular proteins and are involved in a variety of signaling pathways, including cell cycle control, regulation of cell division, apoptosis, antiviral defense, cancer, and more (Boisvert et al., 2007
; Sirri et al., 2008
). For many viruses, trafficking of viral proteins through nucleoli is an important step in their growth cycle, and accordingly, the viral proteins directed to nucleolus are essential for the viral replication in cell culture. For example, herpesvirus and human immunodeficiency virus proteins that are targeted to the nucleolus have been implicated in viral mRNA processing (Hiscox et al., 2010
). Several proteins of adenoviruses and herpesviruses cause redistribution of nucleolar components, reorganization of the nucleolus and interference with nucleolar function, including disruption of rRNA synthesis, processing and trafficking (Hiscox et al., 2010
VACV affects various host cell processes, in particular translation is shut down at least partly through decapping of host mRNAs (Parrish et al., 2007
) and by localization of the translation apparatus within viral factories (Katsafanas and Moss, 2007
). Host transcription is impaired by mechanisms that have not been well characterized (Puckett and Moss, 1983
). Jefferts and Holowczak (Jefferts and Holowczak, 1971
) reported decreased nucleolar RNA synthesis in VACV-infected L cells and detected peptide from 14
C-labeled virions in nucleoli. In our studies, the F16 protein was specifically targeted to the nucleoli of transfected HeLa cells but it had no obvious effect on rRNA synthesis as determined by EU incorporation and click chemistry. In a preliminary experiment, we did not see greater EU labeling of HeLa cell nucleoli in cells infected with the F16 deletion mutant compared to wt virus (TGS, unpublished). Thus, F16 may affect another, perhaps signaling functions of the nucleoli. Further studies of this phenomenon could reveal an additional layer of manipulation of host cell functions by poxviruses.